Post-translational modification of the ?-amino group of proteins has been documented for many decades. However, these modifications are little studied and thought to be constitutive, with few regulatory roles. Recent data from our laboratory challenges these beliefs. We have identified two novel N-terminal modifying enzymes, NRMT1 and NRMT2, and shown that many of their targets are known disease-related proteins (including Retinoblastoma, SET, Tau, ?-catenin, and ataxin- 3). We have constructed the first viable knockout mouse for any N-terminal PTM (NRMT1-/-), which has severe developmental defects. We have also identified the first protein, MYL9, which can be either N-terminally methylated or N-terminally acetylated. According to these data, we aim to demonstrate that N-terminal post- translational modifications are dynamic, serve distinct functional roles, and control many pathways regulating human development and disease. Specific Aim #1 will use our newly generated NRMT1-/- mouse embryonic fibroblasts to determine the functional differences between N-terminal mono- and trimethylation. Specific Aim #2 will use a combination of biochemical assays and mass spectrometry to show for the first time that N-terminal methylation and N-terminal acetylation are interchangeable and differentially regulate protein function. Specific Aim #3 will use a combination of isothermal calorimetry, protein arrays, and SILAC mass spectrometry to determine if N-terminal monomethylation, trimethylation, and acetylation promote different binding partners. These experiments will begin to place N-terminal post-translational modifications in their relevant signaling pathways and reveal how their misregulation leads to developmental defects and tumor formation. Successful completion of these aims will initiate a new field of protein regulation and identify novel regulatory mechanisms for many cellular pathways known to impact human health.